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1 Table S1. Summary of sequence strains, sequencing coverage, an spore viability. 1 Strain Nationality Locality Substrate Isolation ate Sequence strain Coverage Other strain Other Coverage Spore viability11 Notes Reference ZP513 Portugal Lisbon Q. pyrenaica Mar. 5 FM % (787789) 6% (16) mixture of 5 bp an 36 bp reas 4 ZP537 Portugal L. Albufeira Q. faginea May 5 FM156 8% (39718) FM % (7843) 11% (8) No MATa or HMR; ZP537 partially enoreuplicate but not FM194 4 ZP54 Portugal Aagoi Q. pyrenaica Jun. 5 FM157 73% (369665) % (4) reason for sterility unknown; 34 bp reas after trimming "GT" barcoe 4 ZP591 Portugal Cast. Vie Q. pyrenaica Aug. 5 FM19 94% (145954) FM % ( ) 94% (16) Portuguese reference strain; mixture of 3 bp an 36 bp reas (ZP591) 4 ZP594 Portugal A. Dez Q. faginea Sep. 5 FM % ( ) 88% (16) 4 ZP595 Portugal A. Dez Q. faginea Sep. 5 FM % (587661) 9% () 4 ZP6 Portugal Arrábia Q. ilex Oct. 5 FM % (38143) 1% (14) 4 ZP61 Portugal Arrábia Q. ilex (accorn) Oct. 5 FM % (38874) 94% (16) 4 ZP63 Portugal Arrábia Q. ilex (leaf litter) Oct. 5 FM % ( ) 95% () 4 ZP65 Portugal Arrábia Q. faginea Oct. 5 FM16 71% (786785) % (1) No MAT or HML; 34 bp reas after trimming "CT" barcoe 4 ZP67 Portugal Arrábia Q. faginea Oct. 5 FM % (314399) 1% (16) 4 ZP69 Portugal L. Albufeira Q. ilex Dec. 5 FM % (76386) 1% (16) 4 ZP63 Portugal L. Albufeira Q. ilex Dec. 5 FM % (44751) 88% (16) 4 ZP634 Portugal Cast. Vie Q. pyrenaica Jan. 6 FM166 68% (86451) % (8) No MATa or HMR; 34 bp reas after trimming "AT" barcoe 4 IFO18 Japan Mt. Daisen partially ecaye leaf < 199 IFO18 1% (reference) IFO18 67% (7133) 3 3 type strain an originally sequence strain 5 IFO199 Japan Mt. Daisen soil < 5 IFO199 89% (3318) 34 bp reas after trimming "GT" barcoe 6 IFO1991 Japan Mt. Daisen partially ecaye leaf < 5 IFO % (39481) 34 bp reas after trimming "TT" barcoe 6 IFO183 Japan Yakushima Islan partially ecaye leaf < 199 IFO183 86% (85464) very istant subspecies; mixture of 34 bp reas after trimming "CT" barcoe an 36 bp reas 5 W7 Switzerlan Waeenswil Lalleman wine strain < 1995 FM143 NA (635858) known hybri of S. cerevisiae an S. kuriavzevii; 34 bp reas after trimming "AT" barcoe 7, 8 CBS679 < 1913 FM154 NA (75549) % () hybri of S. cerevisiae an S. kuriavzevii; 34 bp reas after trimming "TT" barcoe 9 1 Single spore erivative. Wil erivative, presume iploi. 3 The reference sequence was assume correct an complete when analyzing other strains, an the Solexa ata was use only to estimate the error rate of the assemblies. 4 Sampaio JP, Gonçalves P. 8. Natural populations of Saccharomyces kuriavzevii in Portugal are associate with oak bark an are sympatric with S. cerevisiae an S. paraoxus. Appl Environ Microbiol 74: Kaneko Y, Bano I Reexamination of Saccharomyces bayanus strains by DNA-DNA hybriization an electrophoretic karyotyping. IFO Res Commun 15: Mikata K. Japan National Institute of Technology an Evaluation Biological Resource Center ( 7 Schuetz M, Gafner J Dynamics of the yeast strain population uring spontaneous alcoholic fermentation etermine by CHEF gel electrophoresis. Lett Appl Microbiol 19: González SS, Barrio E, Gafner J, Querol A. 6. Natural hybris from Saccharomyces bayanus an Saccharomyces kuriavzevii in wine fermentations. FEMS Yeast Res 6: Guilliermon A Deposite into CBS culture collection. 1 Coverage shows the percent of calle bases, relative to the IFO18 T reference sequence escribe in the Methos, while the number of non-aapter (no exact 15-mer hits) sequencing reas obtaine is in parentheses. 11 Spore viability shows the percent of viable spores with the number of spores teste in parentheses. 1

2 Table S. Summary of numbers of GAL sites available for various analyses. Gene Pseuogene (bp) 1 Coon (bp) S 3 S 4 Phylogenetics 5 All strains 6 Upstream 7 Coing 8 Downstream 9 YML53C (.,.4) SUR (.4,.1) GAL (.39,.6) AIM (.19,.34) YPL47C (.1,.3) GAL (.49,.71) GYP (.,.3) RPL36B (.,.) KAP (.3,.6) GAL (.83,.48) GAL (.98,.6) GAL (1.6,.3) FUR (.14,.) POA (.1,.9) SIC (.1,.6) EMP (.,.7) GAL (1.4, saturate) SRL (.64, 1.) EMP (.1,.4) 1 Length of pseuogenes in IFO18 T (entire intergenic region between ajacent functional genes). Aligne bp remaining after incluing only fully aligne coons between ZP591, IFO18 T, S. bayanus, S. mikatae, S. paraoxus, an S. cerevisiae. 3 Synonymous sites between ZP591 an IFO18 T (Fig. ). 4 95% confience interval of average number of synonymous ifferences between ZP591 an IFO18 T (low, high) (Fig. ). 5 Number of bp in phylogenetic ata matrix with S. castellii an IFO183 ae (Fig. 1a, S3a). 6 Number of bp in phylogenetic ata matrix with full strain set (Fig. S4). 7 Number of upstream bp aligne between ZP591 an IFO18 T (Fig. S5). 8 Number of coing bp aligne between ZP591 an IFO18 T (Fig. S5). 9 Number of ownstream bp aligne between ZP591 an IFO18 T (Fig. S5). Missing ata was either not calculate or not applicable. Table S3. Constitutive expression of the GAL network in S. cervisiae is eleterious uner non-inucing conitions. Genotype % Raffinose (non-inuce) % Glucose (represse) % Galactose (inuce) GAL8 +. ±.1. ±.1. ±.1 gal ± ± ±.3 Malthusian selection coefficients are reporte ± s.. with N = 1 as escribe in the Methos. Note that mutants lacking the Gal8 co-repressor are very unfit in non-inucing conitions, as previously observe in 5% glycerol by MacLean 1 in a stuy of GAL mutants create by the systematic eletion project. 1 Maclean RC. 7. Pleiotropy an GAL pathway egeneration in yeast. J Evol Biol : Giaever G et al.. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:

3 Table S4. List of strains use in experiments. Strain Species Population Derive from Genotype Use Notes FM19 S. kuriavzevii Portuguese ZP591 MATa/MAT Table S1 1 FM171 S. kuriavzevii Portuguese ZP591 MATa/MAT Table S1 FM197 S. kuriavzevii Japanese IFO18 T MAT ho ::natmx 3 FM198 S. kuriavzevii Japanese IFO18 T MATa ho ::natmx 3 FM119 S. kuriavzevii Portuguese FM171 MATa ho ::kanmx 3 FM111 S. kuriavzevii Portuguese FM171 MAT ho ::kanmx 3 FM1111 S. kuriavzevii hybri FM197/FM119 MATa/MAT ho ::kanmx/ho ::natmx Fig. S1 4 FM111 S. kuriavzevii hybri FM198/FM111 MATa/MAT ho ::kanmx/ho ::natmx Fig. S1 4 FM113 S. kuriavzevii Portuguese FM119 MATa ho ::kanmx ura3-5 FM1131 S. kuriavzevii Portuguese FM111 MAT ho ::kanmx trp1-6 FM114 S. kuriavzevii Portuguese FM113 MATa ho ::kanmx ura3- trp1 ::ScerURA3 + 7 FM1146 S. kuriavzevii Portuguese FM114 MATa ho :kanmx ura3- trp1 ::ScerGAL3 + 8 FM1153 S. kuriavzevii Portuguese FM171/FM1146 MATa/MAT ho ::kanmx/ho + ura3- /URA3 + trp1 ::ScerGAL3 + /TRP1 + 9 FM1157 S. kuriavzevii Portuguese FM113/FM1131 MATa/MAT ho ::kanmx/ho ::kanmx ura3- /URA3 + trp1- /TRP1 + 9 FM1159 S. kuriavzevii Portuguese FM1153 MATa ho ::kanmx ura3- trp1 ::ScerGAL3 + Fig. 3c FM116 S. kuriavzevii Portuguese FM1153 MATa ho ::kanmx ura3- trp1 ::ScerGAL3 + Fig. 3c FM1161 S. kuriavzevii Portuguese FM1153 MATa ho ::kanmx ura3- trp1 ::ScerGAL3 + Fig. 3c FM116 S. kuriavzevii Portuguese FM1153 MATa ho ::kanmx ura3- trp1 ::ScerGAL3 + Fig. 3c FM1163 S. kuriavzevii Portuguese FM1153 MATa ho ::kanmx ura3- trp1 ::ScerGAL3 + Fig. 3c FM1164 S. kuriavzevii Portuguese FM1153 MATa ho ::kanmx ura3- trp1 ::ScerGAL3 + Fig. 3c FM1165 S. kuriavzevii Portuguese FM1153 MATa ho ::kanmx ura3- trp1 ::ScerGAL3 + Fig. 3c FM1166 S. kuriavzevii Portuguese FM1153 MATa ho ::kanmx ura3- trp1 ::ScerGAL3 + Fig. 3c FM1183 S. kuriavzevii Portuguese FM1157 MATa ho ::kanmx ura3- trp1- Fig. 3c FM1184 S. kuriavzevii Portuguese FM1157 MATa ho ::kanmx ura3- trp1- Fig. 3c FM1185 S. kuriavzevii Portuguese FM1157 MATa ho ::kanmx ura3- trp1- Fig. 3c FM1186 S. kuriavzevii Portuguese FM1157 MATa ho ::kanmx ura3- trp1- Fig. 3c FM1187 S. kuriavzevii Portuguese FM1157 MATa ho ::kanmx ura3- trp1- Fig. 3c FM1188 S. kuriavzevii Portuguese FM1157 MATa ho ::kanmx ura3- trp1- Fig. 3c FM1189 S. kuriavzevii Portuguese FM1157 MATa ho ::kanmx ura3- trp1- Fig. 3c FM119 S. kuriavzevii Portuguese FM1157 MATa ho ::kanmx ura3- trp1- Fig. 3c FM18 S. cerevisiae laboratory BY474 MATa ura3- lys- P TDH3-yEGFP-T CYC1 Table S3 1 FM183 S. cerevisiae laboratory BY474 MATa ura3- lys- P TDH3-yBGFP-T CYC1 Table S3 11 FM184 S. cerevisiae laboratory FM18 MATa ura3- lys- P TDH3-yEGFP-T CYC1 GAL8 + Table S3 1 FM185 S. cerevisiae laboratory FM18 MATa ura3- lys- P TDH3-yEGFP-T CYC1 GAL8 + Table S3 1 FM186 S. cerevisiae laboratory FM18 MATa ura3- lys- P TDH3-yEGFP-T CYC1 GAL8 + Table S3 1 FM187 S. cerevisiae laboratory FM18 MATa ura3- lys- P TDH3-yEGFP-T CYC1 gal8- Table S3 13 FM188 S. cerevisiae laboratory FM18 MATa ura3- lys- P TDH3-yEGFP-T CYC1 gal8- Table S3 13 FM189 S. cerevisiae laboratory FM18 MATa ura3- lys- P TDH3-yEGFP-T CYC1 gal8- Table S3 13 FM19 S. cerevisiae laboratory FM18 MATa ura3- lys- P TDH3-yEGFP-T CYC1 GAL3 + Fig. 3c 14 FM191 S. cerevisiae laboratory FM18 MATa ura3- lys- P TDH3-yEGFP-T CYC1 GAL3 + Fig. 3c 14 FM19 S. cerevisiae laboratory FM18 MATa ura3- lys- P TDH3-yEGFP-T CYC1 GAL3 + Fig. 3c 14 FM193 S. cerevisiae laboratory FM18 MATa ura3- lys- P TDH3-yEGFP-T CYC1 gal3- Fig. 3c 15 FM194 S. cerevisiae laboratory FM18 MATa ura3- lys- P TDH3-yEGFP-T CYC1 gal3- Fig. 3c 15 FM195 S. cerevisiae laboratory FM18 MATa ura3- lys- P TDH3-yEGFP-T CYC1 gal3- Fig. 3c 15 FM133 S. kuriavzevii Portuguese FM113 MATa ho ::kanmx ura3- gal8 ::ScerURA FM1334 S. kuriavzevii Portuguese FM113 MATa ho ::kanmx ura3- gal8 ::ScerURA FM1335 S. kuriavzevii Portuguese FM113 MATa ho ::kanmx ura3- gal8 ::ScerURA FM1336 S. kuriavzevii Portuguese FM113 MATa ho ::kanmx ura3- gal8 ::ScerURA FM1337 S. kuriavzevii Portuguese FM113 MATa ho ::kanmx ura3- gal8 ::ScerURA FM1338 S. kuriavzevii Portuguese FM113 MATa ho ::kanmx ura3- gal8 ::ScerURA FM134 S. kuriavzevii Portuguese FM113 MATa ho ::natmx ura3- Fig. 3b 17, 18 FM1343 S. kuriavzevii Portuguese FM113 MATa ho ::natmx ura3- Fig. 3b 17 FM1345 S. kuriavzevii Portuguese FM113 MATa ho ::natmx ura3- Fig. 3b 17 FM1346 S. kuriavzevii Portuguese FM113 MATa ho ::natmx ura3- Fig. 3b 17 FM1348 S. kuriavzevii Portuguese FM113 MATa ho ::natmx ura3- Fig. 3b 17 FM1351 S. kuriavzevii Portuguese FM113 MATa ho ::kanmx ura3- gal8 Fig. 3b 19 FM135 S. kuriavzevii Portuguese FM113 MATa ho ::kanmx ura3- gal8 Fig. 3b 19 FM1353 S. kuriavzevii Portuguese FM113 MATa ho ::kanmx ura3- gal8 Fig. 3b 19 FM1354 S. kuriavzevii Portuguese FM1134 MATa ho ::kanmx ura3- gal8 Fig. 3b 19 FM1355 S. kuriavzevii Portuguese FM1135 MATa ho ::kanmx ura3- gal8 Fig. 3b 19 FM1356 S. kuriavzevii Portuguese FM1135 MATa ho ::kanmx ura3- gal8 Fig. 3b 19 FM1357 S. kuriavzevii Portuguese FM1136 MATa ho ::kanmx ura3- gal8 Fig. 3b 19 FM1358 S. kuriavzevii Portuguese FM1137 MATa ho ::kanmx ura3- gal8 Fig. 3b 19 FM1359 S. kuriavzevii Portuguese FM1137 MATa ho ::kanmx ura3- gal8 Fig. 3b 19 FM136 S. kuriavzevii Portuguese FM1137 MATa ho ::kanmx ura3- gal8 Fig. 3b 19 FM1361 S. kuriavzevii Portuguese FM1138 MATa ho ::kanmx ura3- gal8 Fig. 3b 19 FM136 S. kuriavzevii Portuguese FM1138 MATa ho ::kanmx ura3- gal8 Fig. 3b 19 1 Lab stock of wil ZP591 isolate; Portuguese reference strain. Homozygote of ZP591 erive from a single spore. 3 Heterothallic single spore isolate after eletion of HO. 4 F 1 Japanese/Portuguese strain sporulate to generate F segregants. 5 Start coon to stop coon eletion mae transforming with PCR prouct an selecting against URA Start coon to stop coon eletion mae transforming with PCR prouct an selecting against TRP Start coon to stop coon replacement mae transforming with PCR prouct an selecting for URA3 + plus its intergenic sequences. 8 Start coon to stop coon replacement mae transforming with PCR prouct containing ScerGAL3 + plus its intergenic sequences an selecting against URA Cross use to generate panel of experimental strains. 1 GFP competition strain BFP competition strain 1. 1 GAL8 + re-introuce in triplicate strains to MATa ura3- lys- P TDH3-yEGFP-T CYC1 gal8 ::URA3 + by transforming with PCR prouct an selecting against URA URA3 + remove from MATa ura3- lys- P TDH3-yEGFP-T CYC1 gal8 ::URA3 + by transforming with engineere oligonucleoties an selecting against URA GAL3 + re-introuce in triplicate strains to MATa ura3- lys- P TDH3-yEGFP-T CYC1 gal3 ::URA3 + by transforming with PCR prouct an selecting against URA URA3 + remove from MATa ura3- lys- P TDH3-yEGFP-T CYC1 gal3 ::URA3 + by transforming with engineere oligonucleoties an selecting against URA Start coon to stop coon replacement mae transforming with PCR prouct an selecting for ScerURA3 + plus its intergenic sequences. 17 Drug marker change by selecting for natmx, control competition strains compete against parental FM Competition strain with no etectable efect. 19 Start coon to stop coon eletion mae transforming with PCR prouct an selecting against URA3 +, compete against FM134. Sampaio JP, Gonçalves P. 8. Natural populations of Saccharomyces kuriavzevii in Portugal are associate with oak bark an are sympatric with S. cerevisiae an S. paraoxus. Appl Environ Microbiol. 74: Hittinger CT, Carroll SB. 7. Gene uplication an the aaptive evolution of a classic genetic switch. Nature 449:

4 Figure S1 5 4 Number of F segregants 3 Observe Expecte Number of functional GAL + loci Figure S1. The S. kuriavzevii GAL loci segregate inepenently. Histogram of the number of functional GAL + loci recovere from F segregants from a cross of a Gal + Portuguese strain an a Gal - Japanese strain. Spore viability was high (8%), an alleles at the GAL loci assort inepenently (P =.36). Blue, observe; re, expecte. See attache. 4

5 oi: 1.138/nature8791 Supplementary Information accompanies the paper on. Table S1. Summary of sequence strains, sequencing coverage, an spore viability. See attache. Table S. Summary of numbers of GAL sites available for various analyses. See attache. Table S3. Constitutive expression of the GAL network in S. cerevisiae is eleterious uner non-inucing conitions. See attache. Table S4. List of strains use in experiments. See attache. Figure S1. The S. kuriavzevii GAL loci segregate inepenently. Histogram of the number of functional GAL+ loci recovere from F segregants from a cross of a Gal+ Portuguese strain an a GalJapanese strain. Spore viability was high (8%), an alleles at the GAL loci assort inepenently (P =.36). Blue, observe; re, expecte. See attache. Figure S. The wil isolates of S. kuriavzevii are not hybris. Proportion of hits to each Saccharomyces species (y-axis) for each gene (x-axis, sorte by systematic name in S. cerevisiae) base on Solexa short-rea ata from the Portuguese reference strain (a), a known1 S. cerevisiae/s. kuriavzevii hybri (b), an another S. cerevisiae/s. kuriavzevii hybri not isolate from the wil (c). By efinition, the sum of the proportion of hits from each species is 1. for each gene. Therefore, the closer the value of S. kuriavzevii (blue iamon) is to 1. for a given gene, the more confiently introgression can be exclue. In the two hybri strains, regions are clearly evient where only one species has contribute genetic material, as are likely regions of aneuploiy. Note that Database S1 contains the complete unfiltere ata for all strains, as well as analyses with more liberal mismatch criteria, but no convincing evience of introgression. See attache. Figure S3. Relaxe molecular clock estimation of coalescence of GAL an non-gal loci. Estimation of 5

6 a S. cerevisiae S. paraoxus S. mikatae S. kuriavzevii ZP S. kuriavzevii IFO18 T.581 S. kuriavzevii IFO S. bayanus.447 S. castellii b S. cerevisiae S. paraoxus.85 S. mikatae.44.4 S. kuriavzevii ZP S. kuriavzevii IFO18 T.4.9 S. kuriavzevii IFO S. bayanus c.38 S. castellii ZP591 Portuguese Population IFO18 T Japanese Population.141 IFO183 Figure S3. Relaxe molecular clock estimation of coalescence of GAL an non-gal loci. Estimation of relative timing of coalescence of Saccharomyces GAL genes an pseuogenes (a), an equivalent number of sites selecte ranomly from non-gal genes (b), an an equivalent number of sites selecte ranomly from non-gal genes using all strains of S. kuriavzevii shown in Fig. 1c. Scales show estimate substitutions per site; branch lengths are printe immeiately above or below their respective internoes or above the population whose coalescence they estimate. See attache. 6

7 98/91 S. bayanus S. kuriavzevii S. mikatae 1/1 99/84 99/1 99/1 S. paraoxus 99/1 99/1 S. cerevisiae S. castellii.8 Figure S4. No sequence strains of Saccharomyces support introgression as the source of the functional S. kuriavzevii GAL genes. Phylogeny built from aligne an concatenate GAL genes an pseuogenes from Fig. 1a with strains ae from the impute Q ataset of the Saccharomyces Genome Resequencing Project, which inclues the previously escribe species of Saccharomyces boularii an Saccharomyces cariocanus as strains of S. cerevisiae an S. paraoxus, respectively. Values correspon to Bayesian posterior probabilities an bootstrap values obtaine with maximum likelihoo, respectively. Scales show estimate substitutions per site uner the Bayesian framework. Internoes corresponing to recognize species are arker, while Portuguese (blue) an Japanese (re) lineages are colore. See attache. 7

8 Figure S5 a b GAL8.3 GAL4...1 c.6.1 substitutions per site; branch lengths are printe immeiately above or below their respective internoes or above the population whose coalescence they estimate. See attache. Figure S4. No sequence strains of Saccharomyces support introgression as the source of the functional S. kuriavzevii GAL genes. Phylogeny built from aligne.6 an concatenate GAL genes an pseuogenes GAL7 from Fig. 1a with strains ae from the impute Q ataset of the Saccharomyces Genome.5 GAL1 GAL1 GAL Resequencing Project, which inclues the previously escribe species of Saccharomyces boularii an.4 Saccharomyces cariocanus as strains of S. cerevisiae an S. paraoxus, respectively. Values correspon.3 to Bayesian posterior probabilities an bootstrap values obtaine with maximum likelihoo, respectively.. Scales show estimate substitutions per site uner the Bayesian framework. Internoes corresponing to.1 recognize species are arker, while Portuguese (blue) an Japanese (re) lineages are colore. See attache. Figure S5. The ivergence of the GAL loci contrasts sharply with the rest of the genome at nearly all sites. Sliing winow estimates of pairwise ivergence () between ZP591 an IFO18 T : GAL8 (a), GAL4 (b), GAL7/GAL1/GAL1 (c), an GAL (). GAL coing regions are re an oriente from left to right (except GAL7 an GAL1), while intergenic regions are black. Tick marks on the x-axis represent 1 aligne bps, an a ashe line shows the genome-wie backgroun of.11. Note that nearly all promoters an untranslate regions also possess elevate ivergence levels. See attache. Database S1. Complete supporting ata for Figure S. See Excel file. Database S. Divergence of all genes between Japanese (IFO18 T ) an Portuguese (ZP591) reference strains. See Excel file. 1 Gonzalez, S. S., Barrio, E., Gafner, J. & Querol, A. Natural hybris from Saccharomyces cerevisiae, Saccharomyces bayanus an Saccharomyces kuriavzevii in wine fermentations. FEMS Yeast Res 6, , (6). Liti, G. et al. Population genomics of omestic an wil yeasts. Nature 458, , (9). 8

9 1 Gonzalez, S. S., Barrio, E., Gafner, J. & Querol, A. Natural hybris from Saccharomyces cerevisiae, Saccharomyces bayanus an Saccharomyces kuriavzevii in wine fermentations. FEMS Yeast Res 6, , (6). Liti, G. et al. Population genomics of omestic an wil yeasts. Nature 458, , (9). 9